Detrimental role of dendritic cells during Yersinia enterocolitica infection in vivo.

LUCERO ESTRADA CECILIA STELLA MARYS

Bayreuth

Congreso; Alexander von Humboldt Netzwerktagnung.; 2010

Fundación Alexander von Humboldt

Yersinia enterocolitica blocks T cell priming, required for the control of Y. enterocolitica infection, by inhibiting maturation and antigen uptake as well as by the induction of cell death of bone marrow-derived Denditric Cells (DCs). In the present project the role of DCs during an infection with Y. enterocoliticain vivo using ?DC less mice? (CD11c.DOG mice) compared to C57BL/6 wild type (wt) will be investigated. CD11c.DOG mice are transgenic mice expressing the human diphtheria toxin (DT) receptor under the control of the CD11c promoter. Repetitive application of DT leads to a long term ablation of DCs. One day before the infection 8 ng/g bodyweight of DT will be injected intraperitoneal to CD11c.DOG and wt mice as control. The Y. enterocolitica WAP O:8 strain will be used in concentrations of 1x10e3 CFU/mouse (low dose) and 5x10e4 CFU/mouse (high dose) by i.v. infections. The number of viable bacteria will be counted in spleen and liver after 3h, 1, 3 and 7 days; furthermore, the survival of wt and CD11c.DOG mice will be determined.In previous studies in Dr. Autenrieth?s laboratory it was observed that the CFU of the CD11c.DOG mice was less than that of wt mice 1 and 3 dpi. So, in order to determine the cell population in the spleen during the infection, the cells will be analysed by flow cytometry with different antibodies for macrophages, granulocytes, DCs, T cells, NK cells and B cells, using the same period of time. In order to analyse a proinflammatory immune reaction the following cytokines and chemokines will be detected by Flow Cytometry and ELISA from the cell lysate and sera upon infection of the mice: IL-1, CxCL1/KC, IL-18, IFN and TNF. To determine if there is any difference between the uptake and killing process of Y. enterocolitica by macrophages, granulocytes, and DCs these cells will be isolated from the spleen, treated with the antibiotic gentamicine to kill extracelular bacteria, and afterwards plated on Mueller Hinton (MH) agar to determine the amount of intracellular bacteria. Furthermore, the proliferation of T cells will be determine by adoptive transfer of CFSE-labeled OVA-specific CD4 T cells into CD11c.DOG mice treated with DT (and without DT treatment) and infected with Y. enterocolitica. The results obtained with this project could provide important information of Y. enterocolitica infection mechanism in vivo and host response against this infection.